Pneumocystis pneumonia (PCP) is the most common and life threatening opportunistic infection in AIDS patients and is responsible for extensive morbidity and mortality. The pathogen that causes the infection in humans is Pneumocystis jirovecii and has remained an elusive target due to a lack of in vitro culture systems and animal models. Pneumocystis carinii, the organism that infects mice and not humans, has been used as a surrogate for the human pathogen both in culture and in animal models to evaluate potential treatment options. Trimethoprim/sulfamethoxazole the combination of a dihydrofolate reductase (DHFR) inhibitor trimethoprim and a dihydropteroate synthase (DHPS) inhibitor, sulfamethoxazole is the most effective first- line treatment for PCP. The failure of this option due to adverse reactions to the sulfa component as well as recently identified resistant mutants of the DHPS enzyme and the attending adverse reactions and failure rate of second-line agents have prompted an extensive search for alternate treatment options for this AIDS defining, life threatening infection. We have very recently isolated and characterized the DHFR from the human pathogen, P. jirovecii and showed it to be quite different from P. carinii DHFR with respect to inhibitory activities of therapeutic agents. In addition, we have, for the very first time, identified two compounds that are single digit nanomolar inhibitors of pjDHFR with one compound having a remarkable 2190-fold selectivity for the human pathogen DHFR compared to the human DHFR. We also have evidence from a previous study that compounds of this class efficiently penetrate intact organism. The specific aims of this application are: 1) to synthesize compounds in Series I-XI;2) to evaluate the compounds as inhibitors of pjDHFR and hDHFR;3) to evaluate selected analogs from Aim 2 for cytotoxicity in human embryonic lung fibroblasts;4) to evaluate selected analogs (two to four) on the basis of Aims 2 and 3 in a rat model of P. carinii;and 5) to carry out X-ray crystal structure determination of the two parent compounds along with compounds from this study with pjDHFR and hDHFR to afford a molecular understanding of the structural reasons for the potency and selectivity of the analogs. This study will afford a unique opportunity to determine the structural requirements of potent and selective inhibitors of DHFR from P. jirovecii, the pathogen that causes PCP in humans using exceptional lead analogs that we have identified, that will also help in future analog design. In addition, it will allow, for the first time, a selection of compounds for animal model studies using the DHFR from the human pathogen rather than from a surrogate. We expect the study to also perhaps afford potential compounds, for clinical use against PCP in humans as well as against resistant strains, obtained on the basis of selection that originates with pjDHFR inhibition data that is much more meaningful to the clinical setting. These agents could be used alone or in combination to treat PCP thus providing novel agents against a new target.